Many flaviviruses, including West Nile virus (WNV), Japanese encephalitis virus, tick- borne encephalitis virus and dengue virus, are significant human pathogens. No effective antiviral therapies currently exist for treating individuals with flavivirus infections and flavivirus-induced pathogenesis is not completely understood. Evidence for the existence of two Type 1 IFN- and IRF-3/7-independent cellular backup up-regulation pathways for subsets of interferon stimulated genes (ISG) and for a novel virus- mediated counteraction mechanism directed at suppressing protein production by a subset of antiviral ISGs have been discovered. Initial exploratory studies are proposed to define the mechanisms involved. The specific aims are focused on gaining an initial understanding of: (1) the mechanisms responsible for the up-regulation of a subset of IRF-3/7-independent ISGs by 12 hr after infection in the absence of a canonical Type I interferon signaling pathway in WNV-infected cells and for the different regulatory mechanism required for a second subset of ISGs that show delayed activation in WNV-infected cells; and (2) the mechanism by which protein expression by the second subset of ISGs is suppressed in WNV-infected cells. Under Aim 1, we will map the critical transcription factor binding sites (TFBSs) involved in interferon-independent up-regulation of ISGs in the promoters of model ISGs that are rapidly up-regulated and those that show delayed up-regulation in WNV- infected cells. The functional relevance of the mapped TFBSs will be tested by mutagenesis and by in vitro and in vivo DNA-protein interaction assays. Transcription factors binding to the mapped TFBSs will be identified by MassSpec sequencing of proteins obtained by DNA pull-down. Under Aim 2, the contributions of ISG mRNA stability, cell RNA binding proteins and cell miRNAs in reducing protein levels expressed by a subset of ISGs in WNV-infected cells will be investigated. A detailed understanding of these mechanisms will increase knowledge about the complexity of the host innate response to virus infection and about a novel viral counteraction mechanism. Delineation of the viral counteraction mechanism will provide a new cell target for the development of antiviral therapeutics that may be applicable to a broad spectrum of virus infections. PUBLIC HEALTH RELEVANCE: Many flaviviruses, such as West Nile virus, tick-borne encephalitis virus, and dengue virus, are human pathogens causing significant human morbidity and mortality in ever expanding regions of the world. No effective anti-flaviviral therapies currently exist. We propose to analyze two novel Type 1 interferon- and IRF- 3/7-independent pathways for activating host innate antiviral genes in infected cells and a novel mechanism of viral counteraction of host innate responses recently discovered by our lab. A detailed understanding of these mechanisms will increase knowledge about the host innate response to virus infection and viral virulence.